MICROBES NUTRIENT UP
TAKE WHILE DORMANCY
Common Features of
Quiescent Cells
Carbon
Storage An almost universal property of quiescent cells is
the accumulation of carbon stores, although the chemical structure of the
storage form can differ. During low growth states, the yeast Saccharomyces
cerevisiae accumulates glycogen, trehalose, and triglycerides as the main forms
of metabolizable carbon (Gray et al., 2004).
The
bacterial pathogen Vibrio cholerae accumulates glycogen in preparation for
survival in nutrient-poor environments (Bourassa and Camilli, 2009).
Additionally, many bacteria store fatty acids in the form of triglycerides
(Daniel et al., 2004; Kalscheuer et al., 2007) and wax esters (Sirakova et al.,
2012). Both triglycerides and wax esters also accumulate in plant seeds (Radunz
and Schmid, 2000), indicating that this mode of storage is advantageous for
organisms that represent vastly separated domains of life.
In
addition, linear plastic polymers like polyhydroxyalkanoates and
poly-b-hydroxybutyric acid can serve as a carbon repository in a variety of
bacteria living in the soil and the rhizosphere (Kadouri et al., 2005).
What
is the purpose of carbon storage? The most intuitive answer is that these cells
are simply ‘‘storing nuts for winter,’’ and these nutritional stores can be
rapidly mobilized to fuel growth when environmental conditions improve. This
role has been most clearly demonstrated in the S. cerevisiae cell, where the
trehalose stores that accumulate in stationary cultures are immediately
consumed upon addition of fresh media to fuel rapid regrowth (Shi et al.,
2010).
Glycogen
may serve a similar role in V. cholerae, a bacterium whose life cycle relies on
periodic switches from the nutrient-replete mammalian gut to nutrient poor
aquatic environments (Bourassa and Camilli, 2009). Carbon storage has also been
found to play an important role in remodeling cellular carbon fluxes and
facilitating entry into the quiescent state.
Diverse stresses, such as low oxygen, low pH,
or low iron, all induce a storage response in M. tuberculosis through the
activation of a common sensor-kinase system, DosRST (Bacon et al., 2007; Baek
et al., 2011; Daniel et al., 2011). The DosS sensor likely responds to
alterations in cellular redox state in these contexts (Honaker et al., 2010),
and triggers the synthesis of triglycerides that are stored in large cytosolic
inclusions (Garton et al., 2002). The impact of this response appears to extend
beyond the generation of nutrient stores. That is, disruption of the
triglyceride biosynthesis pathway in M. tuberculosis reverses the growth arrest
that is normally caused by these stresses, but has little effect on the
subsequent recovery of growth when the stress is relieved (Baek et al., 2011).
This inverse relationship between growth and triglyceride production appears to
result from the redirection of acetyl-CoA from the TCA cycle, where it is used
to generate energy during aerobic respiration, into lipid synthesis, where
acetyl CoA serves as a building block for fatty acids. The growth-limiting
effect of carbon storage is unlikely to be restricted to mycobacteria. For
example, S. cerevisiae mutants that are unable to produce glycogen or trehalose
consume more CO2 than the wild-type strain during slow growth (Sillje´ et al.,
1999), indicating higher TCA flux in the absence of carbon storage. The almost
universal propensity of microorganisms to accumulate acetyl CoA-derived carbon
stores under growth-limiting stresses suggests that this may represent a common
strategy for reducing growth and metabolic rate.
Cell
Wall Modification
Virtually all bacteria are surrounded by an
elastic meshwork of peptidoglycan that maintains cellular integrity under
changing environmental conditions. This structure is composed of glycan chains,
consisting of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM),
crosslinked through short peptide moieties. Not surprisingly, the long-term
survival of both spores and quiescent cells depends on specific alterations in
the composition of this structure. For example, in stationary phase cultures,
the Gram-positive bacteria Staphylococcus aureus generates a cell wall that is
structurally different from the peptidoglycan found during exponential phase
growth, in that it contains fewer pentaglycine bridges, which crosslink the
glycan chains, and is significantly thicker (Zhou and Cegelski, 2012).
Similarly,
the level and gradient of crosslinking are important for the formation of
bacterial spores. In the spore peptidoglycan layer of the soil-dwelling
bacteria Bacillus subtilis, the peptide side chains serving as crosslinkers are
completely or partially removed from the NAM residues and replaced by
muramic-dlactam, a specificity determinant for germination autolytic enzymes,
at every second NAM position in the cortex glycan strands. As a consequence,
overall levels of crosslinking are markedly decreased in the spore cortex as
compared to the vegetative cell wall (Atrih et al., 1996). Thus, common
features of the peptidoglycan in both quiescent cells and spores are reduced
crosslinks and increased peptidoglycan mass.
The
regulation of these modifications is likely complex, but recent observations
suggest that extracellular D-amino acids, such as D-methionine and D-leucine,
could play an important role. D-amino acids accumulate to millimolar levels in
the supernatants of stationary phase bacterial culture, where they regulate
cell wall synthetic enzymes and are incorporated into the peptidoglycan
polymer. The increased abundance of D-amino acids in cultures of nongrowing
cells and their ability to alter the osmotic sensitivity of V. cholerae (Lam et
al., 2009) suggests a likely role in remodeling the cell wall for quiescence. During
exponential growth, M. tuberculosis peptidoglycan is crosslinked largely via
linkages between the third and fourth amino acids in the stem peptide, the
chain of amino acids in peptidoglycan that crosslinks adjacent strands (i.e.,
4/3 linkages).
In
addition to its structural roles, cell wall metabolism also appears to play an
important role in generating signals that regulate the germination of spores
and the exit from quiescence.
It
may seem intuitive that RNA and protein synthesis will proceed at negligible
rates in the quiescent cell. Protein turnover increases 5-fold in famished E.
coli cells due to proteases that are produced in early stationary phase.
Indeed,
quiescence in S. cerevisiae is accompanied by a 3- to 5-fold decrease in
overall transcription rate (Choder, 1991), and a 20-fold decrease in protein
synthesis (Fuge et al., 1994). The same analysis has not been performed on nonreplicating
cells, and it remains likely that both initiation and elongation rate slow.
Energetics
and Metabolism during Quiescence
Maintenance
of membrane potential and ATP synthesis is not required for sustaining the
viability of spores, even though a repertoire of ATPases and ATP-dependent
regulatory proteins is utilized during the initiation of germination
(Errington, 2003). In contrast, quiescent bacteria maintain their membrane
potential (Pernthaler and Amann, 2004; Rao et al., 2008), and energy homeostasis
appears to be critical for survival. In nonreplicating M. tuberculosis cells
starved for oxygen or nutrients, ATP levels are maintained at a steady level,
which is only 5-fold lower than replicating cells (Gengenbacher et al., 2010;
Rao et al., 2008). This maintenance of ATP homeostasis is clearly important, as
disruption of the proton motive force or chemical inhibition of the F0F1 ATP
synthase involved in ATP synthesis induces cell death in nutrient-starved or
hypoxic cultures (Rao et al., 2008; Sala et al., 2010). Diverse strategies can
be used to maintain energy homeostasis.
Preservation
of Genome Integrity
Maintaining
genome fidelity when little or no metabolic capacity is available for canonical
DNA repair mechanisms is a challenge faced by both quiescent cells and dormant
spores. One strategy common to both types of cells is altering chromosomal
structure to a more chemically stable form. The chromosome of stationary phase
E. coli assumes an extremely compact structure. A nucleoid-associated protein
called Dps, which is expressed only in stationary phase, mediates
biocrystallization of the nucleoid and protects DNA from damage (Martinez and
Kolter, 1997). This compaction of DNA can be very dynamic as bacteria enter and
exit different growth states.
In
the photosynthetic cyanobacterium, Synechococcus elongates, a circadian clock controlled
mechanism induces periodic chromosome compaction during the night (Smith and
Williams, 2006), and the resulting alterations in DNA supercoiling control
global gene expression patterns (Vijayan et al., 2009). M. tuberculosis might
use a similar mechanism to protect its chromosome.
Truly
dormant spores are not able to actively maintain their chromosome but depend on
the induction of DNA repair systems upon exit from the dormant state. M.
tuberculosis, exit the cell cycle with two chromosomal copies (Wayne, 1977).
Thus, high-fidelity recombinational repair mechanisms, which often dominate in
growing cells, are only available to a subset of quiescent organisms. Despite
the apparent presence of a recombinational template in nonreplicating M.
tuberculosis, this organism still appears to utilize more error-prone repair
systems. For example, error-prone translation polymerases, which replicate past
DNA damage lesions, are important for the survival of slowly growing M.
tuberculosis in chronically infected animals (Boshoff et al., 2003).
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